Molecular characterization of the Cryptosporidium cervine genotype from a sika deer (Cervus nippon Temminck) in Zhengzhou, China and literature review.

Abstract

A total of 124 fecal specimens were collected from four deer farms in Zhengzhou City, China and examined for Cryptosporidium by Sheather's sugar flotation technique. Cryptosporidim oocysts were detected in two 1-year-old sika deer, and one of the two specimens was genotyped by sequence and phylogenetic analyses of the small subunit ribosomal RNA (rRNA) (18S rRNA), 70-kDa heat shock protein (HSP70), actin, and Cryptosporidium oocyst wall protein (COWP) genes. Results obtained suggested that the Cryptosporidium studied belonged to Cryptosporidium cervine genotype, although slight sequence differences were noticed at the three loci. The similarities between this isolate and other Cryptosporidium cervine genotype isolates were 99.1-99.8%, 9.8%, 99.7%, and 100% at the 18S rRNA, HSP70, actin, and COWP loci, respectively. This study is the first report of Cryptosporidium infection in sika deer in China.

Full text

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ORIGINAL PAPER
Molecular characterization of the Cryptosporidium cervine
genotype from a sika deer (Cervus nippon Temminck)
in Zhengzhou, China and literature review
Rongjun Wang & Jinchan Wang & Mingfei Sun &
Hailiang Dang & Yaoyu Feng & Changshen Ning &
Fuchun Jian & Longxian Zhang & Lihua Xiao
Received: 8 May 2008 /Accepted: 27 May 2008
# Springer-Verlag 2008
Abstract A total of 124 fecal specimens were collected from
four deer farms in Zhengzhou City, China and examined for
Cryptosporidium by Sheather’s sugar flotation technique.
Cryptosporidim oocysts were detected in two 1-year-old sika
deer, and one of the two specimens was genotyped by se-
quence and phylogenetic analyses of the small subunit ri-
bosomal RNA (rRNA) (18S rRNA), 70-kDa heat shock
protein (HSP70), actin, and Cryptosporidium oocyst wall
protein (COWP) genes. Results obtained suggested that the
Cryptosporidium studied belonged to Cryptosporidium cer-
vine genotype, although slight sequence differences were
noticed at the three loci. The similarities between this isolate
and other Cryptosporidium cervine genotype isolates were
99.1–99.8%, 9.8%, 99.7%, and 100% at the 18S rRNA,
HSP70, actin, and COWP loci, respectively. This study is the
first report of Cryptosporidium infection in sika deer in China.
Introduction
Cryptosporidiosis is a significant cause of diarrheal dis-
eases in human worldwide, usually by routes of water-
borne or food-borne transmission. So far, at least seven
Cryptosporidium species (C. hominis, C. parvum, C.
meleagridis, C. felis, C. canis, C. muris, and C. suis) and
one genotype (Cryptosporidium cervine genotype) have
been shown to be responsible for human infections (Xiao
et al. 2004).
Cryptosporidium cervine genotype has a wide host and
geographic range. It has been found in sheep in Australia
and USA (Ryan et al. 2005; Santin et al. 2007), an ibex in
China (Karanis et al. 2007), one blesbok, mouflon sheep,
and nyala each in Czech Republic (Ryan et al. 2003),
humans in Canada, England, Slovenia, and USA (Blackburn
et al. 2006; Feltus et al. 2006; Leoni et al. 2006; Ong et al.
2002; Soba et al. 2006; Trotz-Williams et al. 2006), wild
white-tailed deer, eastern gray squirrels, eastern chipmunk,
beaver, red squirrel, oodchuck, deer mice, and raccoon in
New York (Feng et al. 2007; Perz and Le Blancq 2001),
lemurs in USA (da Silva et al. 2003), and water samples in
various geographic locations (Feltus et al. 2006; Jiang et al.
2005; Nichols et al. 2006; Ruecker et al. 2007; Xiao et al.
2000).
There is no report of Cryptosporidium cervine genotype
infection in sika deer in China. In the present study, we
have characterized a Cryptosporidium cervine genotype
isolate from a sika deer (Cervus nippon Temminck) by
sequence and phylogenetic analyses of four genes. Results
suggest that the Chinese sika deer isolate had minor
sequence difference from other known isolates.
Parasitol Res
DOI 10.1007/s00436-008-1069-2
R. Wang:J. Wang:M. Sun:H. Dang:C. Ning:F. Jian:
L. Zhang (*)
The College of Animal Science and Veterinary Medicine,
Henan Agricultural University,
Zhengzhou 450002, People’s Republic of China
e-mail: zhanglx8999@yahoo.com.cn
Y. Feng
School of Resource and Environmental Engineering,
East China University of Science and Technology,
Shanghai 200237, People’s Republic of China
L. Xiao (*)
US Department of Health and Human Services, Division
of Parasitic Diseases, National Center for Zoonotic, Vector-Borne
and Enteric Diseases, Centers for Disease Control and Prevention,
Atlanta, GA 30341, USA
e-mail: lax0@cdc.gov

Page 2

Materials and methods
A total of 124 fecal specimens from sika deer (C. nippon
Temminck), red deer (C. elaphus) and Pere David’s deer
(Elaphurus davidianus) were obtained directly by rectal
collection from four deer farms in Zhengzhou City, China
between 2002 and 2007 (Table 1). No clinical sign was
observed when the fecal specimens were collected. The fecal
specimens were concentrated by the Sheather’s sugar
flotation technique and examined by bright field microscope
under 400× and 1,000×. Two fecal specimens from two 1-
year-old sika deer were Cryptosporidium-positive. One
hundred twenty-four fecal specimens were all stored in
2.5% potassium dichromate at 4°C. After purifying the
oocysts from the two positive specimens by the discontin-
uous density sucrose gradient centrifugation, genomic DNA
was extracted using Mag Extractor-Genome kit (Toyobo,
Osaka, Japan). The DNA was kept at −20°C before it was
used in molecular analysis.
Primers to amplify 18S ribosomal RNA (rRNA;
∼1,700 bp), HSP70 (∼1,950 bp), actin (∼1,066 bp), and
Cryptosporidium oocyst wall protein (COWP; ∼530 bp)
genes were adopted from previous studies (Xiao et al.
2004). Polymerase chain reaction (PCR) products of the
actin gene were cloned, and four recombinant plasmids
were sequenced by TaKaRa Biotechnology Co. Ltd
(Dalian, China) with an ABI PRISM™ 3730 XL DNA
Analyzer (Applied Biosystems, USA) using the Big Dye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosys-
tems). PCR products of the 18S rRNA, HSP70, and COWP
genes were sequenced directly without cloning. The sequen-
ces were deposited in GenBank under accession numbers
DQ898159, DQ898163, EF030519, and EF030518. Phylo-
genetic analyses were performed using the software PHYLIP
version 3.67 (Felsenstein 1989).
Table 1 Deer specimens examined for Cryptosporidium
Animal species Age
group
No.
examined
No. of positive
specimens
Sika deer 4 months
1 year
3 years
5 years
3 years
10 years
2 years
18
30
21
14
0
2
0
0
0
0
0
Red deer5
3
Pere David’s deer33
Fig. 1 Phylogenetic relation-
ship of Cryptosporidium para-
sites inferred by neighbor-
joining analysis of the 18S
rRNA gene based on Kimura
two-parameter model. Bootstrap
values (in percentage) above
50% from 1,000 pseudo-repli-
cates are shown for both the
neighbor-joining (the first value)
and maximum parsimony anal-
yses (the second value). Low =
node with bootstrap values low-
er than 50%. Scale bar indicates
an evolutionary distance of 0.1
substitutions per site in the
sequence
Table 2 Reported cases of human infections with the Cryptosporid-
ium cervine genotype
LocationNo. of
positive
samples
Reference
British Columbia, Canada
Ontario, Canada
Ohio, USA
Wisconsin, USA
England, UK
England and Wales, UK
Ljubljana, Slovenia
Lima, Peru
9
1
1
1
1
6
1
1
Ong et al. (2002)
Trotz-Williams et al. (2006)
Blackburn et al. (2006)
Feltus et al. (2006)
Leoni et al. (2006)
Nichols et al. (2006)
Soba et al. (2006)
Xiao and Cama, unpublished
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Results and discussion
Sequences of the 18S rRNA, HSP70, actin, and COWP
genes were obtained from one of the two Cryptosporidium-
positive specimens (another specimen failed to amplify the
specific fragment of four genes studied). They were aligned
with sequences from other known Cryptosporidium species
and genotypes.
In the 18S rRNA gene, there were two to seven nucleotide
differences out of 667 bp with other Cryptosporidium
cervine genotype isolates (EF362480, EF613340,
AF262328, DQ640639, EF641017, AY737593, AY737592,
DQ19250, EF641018, DQ640640, EF362481, and
EF362479). The similarity (99.1%) between this sika deer
isolate and the Chinese ibex isolate (EF613340) was the
lowest, and the highest similarity (99.8%) was observed with
a USA water sample isolate (EF641018). The similarities
with other USA water sample, sheep isolates, and Canada
human isolates varied from 99.2% to 99.7%. Five copies of
the 18S rRNA gene are present in Cryptosporidium genome,
and previous studies have suggested that there is slight
sequence heterogeneity in some of these copies (Xiao et al.
1999). Therefore, some of the sequence differences could be
due to variations among different copies of the rRNA gene.
In the HSP70 gene, comparing to DQ389176, two
nucleotide changes of T to C and one change of A to C
were observed at nucleotides 594 and 1062 and nucleotide
1809, respectively. The sequence similarity between the
two was 98.8%. This might be due to the single nucleotide
polymorphy (SNP) of HSP70 gene. For example, three
SNPs were present between C. hominis sequences
AF221535 and XM_661662. Similarly, in the actin gene,
there were two nucleotide changes between the current
isolate and DQ389175 at nucleotide 322 and 580, with a
sequence similarity of 99.7%. Further comparison was
limited because of the lack of more Cryptosporidium
cervine genotype actin sequences. On the contrary, no
sequence difference was observed in the COWP gene.
Phylogenetic trees were constructed, and two methods
(neighbor-joining and parsimony) produced similar topolo-
Table 3 Reported natural infections of the Cryptosporidium cervine genotype in animals
AnimalsLocation No. of positive samples Reference
Lemurs
Sheep
Sheep
Ibex
Blesbok
White-tailed deer
Sika deer
Mouflon sheep
Nyala
Eastern gray squirrel
Eastern gray squirrel
Eastern chipmunk
Eastern chipmunk
Red squirrel
Red squirrel
Woodchuck
Beaver
Deer mice
Raccoon
North Carolina, USA
Maryland, USA
Western Australia
Qinghai, China
Prague, Czech Republic
New York, USA
Henan, China
Prague, Czech Republic
New York, USA
New York, USA
New York, USA
New York, USA
New York, USA
New York, USA
New York, USA
New York, USA
New York, USA
New York, USA
New York, USA
7da Silva et al. (2003)
Santin et al. (2007)
Ryan et al. (2005)
Karanis et al. (2007)
Ryan et al. (2003)
Perz and Le Blancq (2001)
DQ898159
Ryan et al. (2003)
Ryan et al. (2003)
Feng et al. (2007)
Ziegler et al. (2007)
Feng et al. (2007)
Ziegler et al. (2007)
Feng et al. (2007)
Ziegler et al. (2007)
Feng et al. (2007)
Feng et al. (2007)
Feng et al. (2007)
Feng et al. (2007)
28
33
1
1
4
1
1
1
3
1
2
3
1
2
1
1
1
1
Table 4 The occurrence of the Cryptosporidium cervine genotype in the environment
Sample typeLocationNo. of positive samplesReference
Source water
Source water
Storm water
Raw wastewater
Raw wastewater
Raw wastewater
Stream sediment
Ontario, Canada
Maryland, USA
New York, USA
Milwaukee, USA
Milwaukee, USA
Tunis, Tunisia
New York, USA
1
2
Ruecker et al. (2007)
Xiao et al., unpublished
Xiao et al. (2000); Jiang et al. (2005)
Zhou et al. (2003)
Xiao et al. (2006a)
Xiao and Ben Ayed, unpublished
Xiao et al., unpublished
63
6
9
1
1
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gy for each of the four loci. In each of the analysis,
sequence from sika deer grouped together with other
Cryptosporidium cervine genotype isolates (a neighbor-
joining tree of the 18S rRNA gene is showed in Fig. 1).
Cryptosporidium cervine genotype is the only species/
genotype with noticeable broad host range (Tables 2 and 3).
Since its initial finding in storm runoff in a wilderness area,
it has been found in domestic and wild ruminants (sheep,
mouflon sheep, ibex, blesbok, nyala, white-tailed deer, and
Pere David’s deer), rodents (squirrels, chipmunks, wood-
chucks, beavers, and deer mice), carnivores (raccoons), and
primates (lemurs and humans; Xiao et al. 2000; Perz and Le
Blancq 2001; Ong et al. 2002; da Silva et al. 2003; Ryan et
al. 2003, 2005; Blackburn et al. 2006; Feltus et al. 2006;
Leoni et al. 2006; Nichols et al. 2006; Soba et al. 2006;
Trotz-Williams et al. 2006; Feng et al. 2007; Karanis et al.
2007). The generalist nature of the host specificity of the
parasite and habitat sharing are probably responsible for the
wide occurrence of the cervine genotype in animals.
According to available data, the cervine genotype has been
found in various environmental samples such as source water,
stream storm runoff, stream sediment, and raw wastewater in
several countries (Table 4). In studies conducted in wilder-
ness areas of the New York City drinking water supply
watershed, the cervine genotype was the most common
Cryptosporidium species/genotype found in river/stream
water (Xiao et al. 2000, 2006b; Jiang et al. 2005) and was
the only year-round Cryptosporidium in storm water (Xiao et
al. 2000, 2006b; Jiang et al. 2005). The finding of the cervine
genotype in these areas suggest the potential for spreading
among wildlife species that routinely drink from streams.
An increasing number of human cases have been
associated with the Cryptosporidium cervine genotype
(Table 2). It has been reported in ten patients in Canada,
seven in the UK, three in the USA, and one in Slovenia
(Ong et al. 2002; Blackburn et al. 2006; Feltus et al. 2006;
Leoni et al. 2006; Nichols et al. 2006; Soba et al. 2006;
Trotz-Williams et al. 2006). Likewise, the increasing
number of humans infected with the cervine genotype
might be related to its wide range of mammalian hosts
(Feng et al. 2007). Almost all the human cases are reported
in industrialized nations where it ranked number four in
Cryptosporidium spp. in humans (more human cases have
been attributed to the cervine genotype than C. canis in
industrialized nations), indicating that zoonotic transmis-
sion is probably responsible for most of the cases with
cervine genotype. However, it has been recently seen in one
child in a shantytown in Lima, Peru where cryptosporidi-
osis is known to be transmitted main via the anthroponotic
route (Xiao and Cama unpublished).
In this study, four deer farms were screened for Crypto-
sporidium oocysts for 5 years, but only two fecal specimens
were positive, with an average infection rate of 1.61%
(2/124). The low infection rate suggests that deer studied
here are not very susceptible to Cryptosporidium and are not
an important reservoir of cryptosporidiosis. Moreover, the
four farms are all far from the drinking water supply system.
Therefore, the deer populations on farms may be not a major
source of cryptosporidiosis in humans in Zhengzhou region.
This study broadens the host and geographic ranges of
the Cryptosporidium cervine genotype and provides new
sequence data. In conjunction with previous reports, it is
expected that the Cryptosporidium cervine genotype will be
named as a new species in future.
Acknowledgments
Innovation Project for Prominent University Research Talents (num-
ber 2004KYCX002).
This study was supported in part by Henan
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